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Background The use of fluorescent proteins to label tumors is revolutionizing cancer research, enabling imaging of both primary and metastatic lesions, which is important for diagnosis, staging, and therapy. This report describes the use of fluorescence laparoscopy to image green fluorescent protein (GFP)-expressing tumors in an orthotopic mouse model of human pancreatic cancer. Methods The orthotopic mouse model of human pancreatic cancer was established by injecting GFP-expressing MiaPaCa-2 human pancreatic cancer cells into the pancreas of 6-week-old female athymic mice. On postoperative day 14, diagnostic laparoscopy using both white and fluorescent light was performed. A standard laparoscopic system was modified by placing a 480-nm short-pass excitation filter between the light cable and the laparoscope in addition to using a 2-mm-thick emission filter. A camera was used that allowed variable exposure time and gain setting. For mouse laparoscopy, a 3-mm 0° laparoscope was used. The mouse’s abdomen was gently insufflated to 2 mm Hg via a 22-gauge angiocatheter. After laparoscopy, the animals were sacrificed, and the tumors were collected and processed for histologic review. The experiments were performed in triplicate. Results Fluorescence laparoscopy enabled rapid imaging of the brightly fluorescent tumor in the pancreatic body. Use of the proper filters enabled simultaneous visualization of the tumor and the surrounding structures with minimal autofluorescence. Fluorescence laparoscopy thus allowed exact localization of the tumor, eliminating the need to switch back and forth between white and fluorescence lighting, under which the background usually is so darkened that it is difficult to maintain spatial orientation. Conclusion The use of fluorescence laparoscopy permits the facile, real-time imaging and localization of tumors labeled with fluorescent proteins. The results described in this report should have important clinical potential.